1. Field of the Invention
The present invention relates to an integrated nucleic acid test cartridge capable of performing amplification based on temperature cycling and isothermal methods. Furthermore, it relates to devices and methods for receiving a sample suspected of containing a nucleic acid target, performing amplification and transferring an amplicon for detection. The amplification cartridge can be equipped with a sensing means including at least optical and electrochemical sensors. The cartridge can perform various methods of amplification including, but not limited to, polymerase chain reaction, rolling circle amplification and strand displacement amplification. The amplification device also has the ability to function with a portable power supply or means therefor.
2. Background Information
Applications of nucleic acid testing are broad. The majority of conventional commercial testing relates to infectious diseases including Chlamydia, gonorrhea, hepatitis and human immunodeficiency virus (HIV) viral load; genetic diseases including cystic fibrosis; coagulation and hematology factors including hemochromatosis; and cancer including genes for breast cancer. Other areas of interest include forensics and paternity testing, cardiovascular diseases and drug resistance screening, termed pharmacogenomics. The majority of testing currently occurs in centralized laboratories using non-portable and operationally complex instruments. Conventionally, tests generally require highly skilled individuals to perform the assays. As a result, the time taken between obtaining a sample suspected of containing a specific nucleic acid fragment and determining its presence or absence is often several hours and even days. However, as with other kinds of blood tests, physicians and scientists often require results more quickly and that are obtainable in a convenient user-friendly format. Consequently, there is a need for a portable analysis system capable of performing nucleic acid testing quickly and conveniently.
Methods of extracting nucleic acids from cells are well known to those skilled in the art. A cell wall can be weakened by a variety of methods, permitting the nucleic acids to extrude from the cell and permitting its further purification and analysis. The specific method of nucleic acid extraction is dependent on the type of nucleic acid to be isolated, the type of cell, and the specific application used to analyze the nucleic acid. Many methods of isolating DNA are known to those skilled in the art, as described in, for example, the general reference Sambrook and Russell, 2001, “Molecular Cloning: A Laboratory Manual,” pages 5.40-5.48, 8.1-8.24, A1.17-A1.19, and A1.25-A1.27. For example, conventional techniques can include chemically-impregnated and dehydrated solid-substrates for the extraction and isolation of DNA from bodily fluids that employ lytic salts and detergents and that contain additional reagents for long-term storage of DNA samples, as described in, for example, U.S. Pat. No. 5,807,527 (detailing FTA paper), and U.S. Pat. No. 6,168,922 (detailing Isocard Paper). Conventional techniques can also include particle separation methods, such as those described in, for example, U.S. Reissue Pat. No. RE37,891.
Several methods and apparatuses for amplification of nucleic acid are known to those of ordinary skill in the art. It is known that Polymerase Chain Reaction (PCR) is inhibited by a number of proteins and other contaminants that follow through during the standard methods of purification of genomic DNA from a number of types of tissue samples. It is known that additional steps of organic extraction with phenol, chloroform and ether or column chromatography or gradient CsCl ultracentrifugation can be performed to remove PCR inhibitors in genomic DNA samples from blood. However, these steps add time, complexity and cost. Such complexity has limited development of a simple disposable cartridge useful for nucleic acid analysis. Therefore, the development of new, simple methods to overcome inhibitors found in nucleic acid samples used for nucleic acid amplification processes is desirable.
Nucleic acid hybridization is used to detect discernible characteristics about target nucleic acid molecules. Techniques like the “Southern analysis” are well known to those skilled in the art. Target nucleic acids are electrophoretically separated, then bound to a membrane. Labeled probe molecules are then permitted to hybridize to the nucleic acids bound to the membrane using techniques well known in the art. This method is limited, however, because the sensitivity of detection is dependent on the amount of target material and the specific activity of the probe, and, in the example of a radioactively labeled probe, the time of exposure of the signal to the detection device can be increased. Alternatively, as the probe's specific activity may be fixed, to improve the sensitivity of these assays, methods of amplifying nucleic acids are employed. Two basic strategies are employed for nucleic acid amplification techniques; either the number of target copies is amplified, which in turn increases the sensitivity of detection, or the presence of the nucleic acid is used to increase a signal generated for detection. Examples of the first approach include polymerase chain reaction (PCR), rolling circle (as described in, for example, U.S. Pat. No. 5,854,033), and nucleic acid system based amplification (NASBA). Examples of the second include cycling probe reaction, termed CPR (as described in, for example, U.S. Pat. Nos. 4,876,187 and 5,660,988) and SNPase assays, e.g., the Mismatch Identification DNA Analysis System (as described in, for example, U.S. Pat. Nos. 5,656,430 and 5,763,178). More recently, a strategy for performing the polymerase chain reaction isothermally has been described by Vincent et al., 2004, EMBO Reports, vol 5(8), and is described in, for example, U.S. Application Publication No. 2004/0058378. A DNA helicase enzyme is used to overcome the limitations of heating a sample to perform PCR DNA amplification.
The PCR reaction is well known to those skilled in the art and was originally described in U.S. Pat. No. 4,683,195. The process involves denaturing nucleic acid, a hybridization step and an extension step in repeated cycles, and is performed by varying the temperature of the nucleic acid sample and reagents. This process of subjecting the samples to different temperatures can be effected by placing tubes into different temperature water baths, or by using Peltier-based devices capable of generating heating and cooling, dependent on the direction of the electrical current, as described in, for example, U.S. Pat. Nos. 5,333,675 and 5,656,493. Many commercial temperature cycling devices are available, sold by, for example, Perkin Elmer (Wellesley, Mass.), Applied Biosystems (Foster City, Calif.), and Eppendorf (Hamburg, Germany). As these devices are generally large and heavy, they are not generally amenable to use in non-laboratory environments, such as, for example, at the point-of-care of a patient.
Microfabricated chamber structures for performing the polymerase chain reaction have been described in, for example, U.S. Pat. No. 5,639,423. A device for performing the polymerase chain reaction is described in, for example, U.S. Pat. No. 5,645,801 that has an amplification chamber that can be mated to a chamber for detection. For example, U.S. Pat. No. 5,939,312 describes a miniaturized multi-chamber polymerase chain reaction device. U.S. Pat. No. 6,054,277 describes a silicon-based miniaturized genetic testing platform for amplification and detection. A polymer-based heating component for amplification reactions is described in, for example, U.S. Pat. No. 6,436,355. For example, U.S. Pat. No. 6,303,288 describes an amplification and detection system with a rupturable pouch containing reagents for amplification. U.S. Pat. No. 6,372,484 describes an apparatus for performing the polymerase chain reaction and subsequent capillary electrophoretic separation and detection in an integrated device.
There are several nucleic acid amplification technologies that differ from the PCR reaction in that the reaction is run at a single temperature. These isothermal methods include, for example, the cycling probe reaction, strand displacement, INVADER™ (Third Wave Technologies Inc., Madison, Wis.), SNPase, rolling circle reaction, and NASBA. For example, U.S. Pat. No. 6,379,929 describes a device for performing an isothermal nucleic acid amplification reaction.
A microfluidic biochemical analysis system with flexible valve ports and with pneumatic actuation is described in, for example, Anderson et al., Transducers '97, pages 477-80; 1997 International Conference on Solid-State Sensors and Actuators, Chicago, Jun. 16-19, 1997. A fully integrated PCR-capillary electrophoresis microsystem for DNA analysis is described in, for example, Lagally et al., Lab on a Chip, 1, 102-7, 2001. A method of non-contact infrared-mediated thermocycling for efficient PCR amplification of DNA in nanoliter volumes is described in, for example, Huhmer and Landers, Analytical Chemistry 72, 5507-12, 2000. A single molecule DNA amplification and analysis microfluidic device with a thermocouple and valve manifold with pneumatic connections is described in Lagally et al., Analytical Chemistry 73, 565-70, 2001.
The polymerase chain reaction (PCR) is based on the ability of a DNA polymerase enzyme to exhibit several core features that include its ability to use a primer sequence with a 3′-hydroxyl group and a DNA template sequence and to extend a newly synthesized strand of DNA using the template strand, as is well known to those skilled in the art. In addition, DNA polymerases used in the PCR reaction must be able to withstand high temperatures (e.g., 90 to 99° C.) used to denature double stranded DNA templates, as well as be less active at lower temperatures (e.g., 40 to 60° C.) at which DNA primers hybridize to the DNA template. Furthermore, it is necessary to have optimal DNA synthesis at a temperature at or above to the hybridization temperature (e.g., 60 to 80° C.).
Zhang et al. (2003, Laboratory Investigation, vol 83(8):1147) describe the use of a terminal phosphorothioate bond to overcome the limitations of DNA polymerases used for 3′-5′ exonuclease activity. The phosphorothioate bond is not cleaved by 3′-5′ exonucleases. This prevents DNA polymerases with 3′-5′ exonuclease activities from removing the terminal mismatch and proceeding with DNA elongation, alleviating the lack of discrimination observed with normal DNA.
Another characteristic of DNA polymerases is their elongation rate. Takagi et al. (1997, Applied and Environmental Microbiology, vol 63(11): 4504) describes that Pyrococcus sp. Strain KOD1 (now Thermococcus kodakaraensis KOD1), Pyrococcus furiosus, Deep Vent (New England Biolabs, Beverly, Mass.), and Thermus aquaticus have elongation rates of 106 to 138, 25, 23 and 61 bases/second, respectively. The processivity rates of these enzymes are also described, and behave similarly to the elongation rates. Clearly, Thermococcus kodakaerensis KOD1 has much higher elongation and processivity rates compared to the other well known enzymes that would make this enzyme beneficial in applications where sensitivity and speed are an issue. Further, Thermococcus kodakaerensis KOD 1 possesses an exonuclease activity that would be detrimental for use in a 3′-allele specific primer extention assay used for SNP analysis.
Conventional detection methods for the final step in a nucleic acid analysis are well known in the art, and include sandwich-type capture methods based on radioactivity, colorimetry, fluorescence, fluorescence resonance energy transfer (FRET) and electrochemistry. For example, jointly-owned U.S. Pat. No. 5,063,081 (the '081 patent) covers a sensor for nucleic acid detection. The sensor has a permselective layer over an electrode and a proteinaceous patterned layer with an immobilized capture oligonucleotide. The oligonucleotide can be a polynucleotide, DNA, RNA, active fragments or subunits or single strands thereof. Coupling means for immobilizing nucleic acids are described along with methods where an immobilized nucleic acid probe binds to a complimentary target sequence in a sample. Detection is preferably electrochemical and is based on a labeled probe that also binds to a different region of the target. Alternatively, an immobilized antibody to the hybrid formed by a probe and polynucleotide sequence can be used along with DNA binding proteins. The '081 patent incorporates by reference the jointly owned U.S. Pat. No. 5,096,669 that is directed to a single-use cartridge for performing assays in a sample using sensors. These sensors can be of the type described in the '081 patent.
Other divisional patents related to the '081 patent include, for example, U.S. Pat. No. 5,200,051 that is directed to a method of making a plurality of sensors with a permselective membrane coated with a ligand receptor that can be a nucleic component. For example, U.S. Pat. No. 5,554,339 is directed to microdispensing, where a nucleic acid component is incorporated into a film-forming latex or a proteinaceous photoformable matrix for dispensing. U.S. Pat. No. 5,466,575 is directed to methods for making sensors with the nucleic component incorporated into a film-forming latex or a proteinaceous photoformable matrix. U.S. Pat. No. 5,837,466 is directed to methods for assaying a ligand using the sensor components including nucleic components. For example, a quantitative oligonucleotide assay is described where the target binds to a receptor on the sensor and is also bound by a labeled probe. The label is capable of generating a signal that is detected by the sensor, e.g., an electrochemical sensor. For example, U.S. Pat. No. 5,837,454 is directed to a method of making a plurality of sensors with a permselective membrane coated with a ligand receptor that can be a nucleic component. Finally, jointly-owned U.S. Pat. No. 5,447,440 is directed to a coagulation affinity-based assay applicable to nucleotides, oligonucleotides or polynucleotides. Each of the aforementioned jointly-owned patents are incorporated by reference herein in their entireties.
It is noteworthy that jointly-owned U.S. Pat. No. 5,609,824 teaches a thermostated chip for use within a disposable cartridge applicable to thermostating a sample, e.g., blood, to 37° C. Jointly-owned U.S. Pat. No. 6,750,053 and U.S. Application Publication No. 2003/0170881 address functional fluidic elements of a disposable cartridge relevant to various tests including DNA analyses. These additional jointly-owned patents and applications are incorporated by reference herein in their entireties.
Several other patents address electrochemical detection of nucleic acids. For example, U.S. Pat. No. 4,840,893 teaches detection with an enzyme label that uses a mediator, e.g., ferrocene. U.S. Pat. No. 6,391,558 teaches single stranded DNA on the electrode that binds to a target, where a reporter group is detected by the electrode towards the end of a voltage pulse and uses gold particles on the electrode and biotin immobilization. For example, U.S. Pat. No. 6,346,387 is directed to another mediator approach, but with a membrane layer over the electrode through which a transition metal mediator can pass. U.S. Pat. No. 5,945,286 is based on electrochemistry with intercalating molecules. For example, U.S. Pat. No. 6,197,508 teaches annealing single strands of nucleic acid to form double strands using a negative voltage followed by a positive voltage. Similar patents include, for example, U.S. Pat. Nos. 5,814,450, 5,824,477, 5,607,832, and 5,527,670 that teach electrochemical denaturation of double stranded DNA. U.S. Pat. Nos. 5,952,172 and 6,277,576 teach DNA directly labeled with a redox group.
Several patents address devising cartridge-based features or devices for performing nucleic acid analyses. Such patents include, for example, a denaturing device described in U.S. Pat. No. 6,485,915, an integrated fluid manipulation cartridge described in U.S. Pat. No. 6,440,725, a microfluidic system described in U.S. Pat. No. 5,976,336 and a microchip for separation and amplification described U.S. Pat. No. 6,589,742.
Based on the forgoing description, there remains a need for a convenient and portable analysis system capable of performing nucleic acid amplification and testing.